Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

Cytokinin treatment of embryos inhibits the synthesis of chloroplast proteins in Norway spruce

A pulse treatment of Norway spruce (Picea abies (L.) Karst) embryos with the cytokinin N⁶-benzyladenine induces the formation of adventitious buds from subepidermal cells in the hypocotyl and cotyledons. In addition the treatment also inhibits elongation growth, a key process during germination. In this report we demonstrate that these effects on development of the plant are associated with a suppression of the accumulation of several major chloroplast proteins during germination. These proteins include the large subunit of ribulose bisphosphate/carboxylase oxygenase, two subunits of the chloroplast ATPase, protochlorophyllide reductase and a 23000-M_r component of photosystem II. For two nuclear-encoded proteins, the small subunit of ribulose bisphosphate carboxylase/oxygenase and the light-harvesting chlorophyll a/b-binding protein, a corresponding suppression of the increase in the steady-state amounts of mRNA is recorded. The suppression of chloroplast protein synthesis is consistant with the previously documented delay in greening that results from cytokinin treatment, but the effect is opposite to that found in other plants, where cytokinins promote the synthesis of chloroplast proteins, and stimulate chloroplast biogenesis. We believe that this difference is explained by the cytokinin primarily suppressing organ development, and a strict dependance of chloroplast biogenesis on the developmental state of the organs.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CF1 coupling-factor 1 of chloroplast ATPase - LHCP light-harvesting chlorophyll a/b-binding protein - LSU large subunit of Rubisco - NADPH-protochlorophyllide oxidoreductase Pchlide reductase - SDS sodium dodecyl sulfate - SSU small subunit of Rubisco We thank K. Hutchison (Dept. of Biochemistry, University of Maine, Orono, Maine, USA) and P. Gustafsson (Dept. of Plant Physiology, University of Umeå, Sweden) for providing the Larix and Pinus clones, and M. Ryberg (Dept. of Plant Physiology, University of Göteborg, Sweden), R. Ölmüller (Botanisches Institut, Universität München, FRG) and W. Lockau (Institut für Botanik, Universität Regensburg, FRG), for the gift of antisera towards Pchlide reductase, RuBPCase and LHCP, and ATPase, respectively. Supported by the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Sciences Research Council.     

Isolation and Purification of Mitochondrial Mn-Superoxide Dismutase from the Gymnosperm Pinus sylvestris L.

Manganese superoxide dismutase (Mn-SOD; EC 1.15.1.1 [EC] ) was purifiedfrom germinating seeds of Scots pine (Pinus sylvestris L.) 3days after the start of imbibition. The purification scheduleincluded (NH₄)₂SO₄ fractionation, anion-exchange and hydrophobic-interactionchromatographies and chromatofocusing. Purified Mn-SOD had anapparent specific activity of 4,130 McCord-Fridovich units (mgprotein)^–1. The molecular mass of the holoenzyme was estimatedto be 91 kDa by size-exclusion chromatography, and a molecularmass of 23 kDa was determined by SDS-PAGE. However, isoelectricfocusing demonstrated that the purified enzyme consisted ofthree similarly migrating isoforms, with isoelectric pointsof approximately 6.5. NH₂-terminal amino acid sequencing ofpurified Mn-SOD revealed no differences among the three isoforms.The comparison of the first 32 NH₂-terminal amino acids withsequences of NH₂-terminal amino acids of Mn-SODs from angiospermsreflected the phylogenetic distances between Scots pine, whichis a gymnosperm, and angiospermic species. Cell fractionationsuggested the mitochondrial localization of Mn-SODs and no evidencefor glyoxysomal localization was found. Mn-SOD activity wasabsent from dry seeds. It was detectable at a considerable levelafter imbibition for 24 h, and it was again absent from 3-week-oldseedlings. (Received February 8, 1994; Accepted May 24, 1994)     

AXanthomonas maltophilia isolate tolerating up to 1% sodium azide in Tris/HCl buffer

A bacterium tolerating up to 1% NaN₃ found as a contaminant of Sephadex colums being run with Tris/HCl buffer, was identified asXanthomonas maltophilia. It had low nutrient requirements, was strongly proteolytic and interfered with Sephadex columns run with Tris/HCl buffers.The authors are with the Department of Plant Pathology, Swedish University of Agricultural Sciences, Box 7044, S-750 07 Uppsala, Sweden;     

Maize embryo germination

The cell-cycle progression of germinating embryos of maize (Zea mays L.) was studied from 0 to 72 h after the start of imbibition using DNA flow cytometry on isolated nuclei, and analyses of thymidine kinase activity, histone biosynthesis and levels of proliferating cell nulcear antigen (PCNA). At the start of germination, 75% of the cells were in G1, but this population had decreased to 25% by 72 h. The concomitant increase of cells in S-phase did not occur continuously, but stepwise, indicating that during germination most of the cells enter S-phase as a partially synchronized population. Within the initial 60 h of embryo germination the cells passed through one S-phase; the start and duration of this period of replicative DNA synthesis was further substantiated by the analysis of S-phase-associated events, the biosynthesis of core histones and the enzyme activity of thymidine kinase, which both began to increase at about 12 h after the start of differentiation. Thymidine kinase fluctuated periodically during germination with a transient maximum at 30 h and a second peak at 72 h; histone biosynthesis was not detectable until 12 h after the start of germination. The levels of PCNA protein closely resembled the pattern of thymidine kinase during germination. Together with the cytometric data this allows a clear assignment of cell cycle events to different times of embryo differentiation.Abbreviation PCNA proliferating-cell nuclear antigen Dedicated to Prof. Walter Larcher on the occasion of his 65th birthdayThe authors wish to thank Prof. G. Mikuz (Department of Pathology, University of Innsbruck, Austria) and Prof. G. Stöffler (Department of Microbiology, University of Innsbruck, Austria) for their interest and support. The technical assistance of Mrs. R. Gantschnig is gratefully acknowledged. E.I. Georgieva was recipient of short-term fellowships from the Austrian Academy of Sciences, the Austrian Forschungsgemeinschaft and the Austrian Akademischer Austauschdienst. G. López-Rodas was recipient of a postdoctoral fellowship from the Programa sectorial de Formación de Profesorado y Personal investigador del Ministerio de Educación y Ciencia (Spain). This work was supported in part by Grant SO6011 (to P.L.) from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung and the Dr. Legerlotz-Foundation.     

Both chloroplastic and cytosolic phosphofructoaldolase isozymes are present in the pea leaf nucleus

Summary. In higher plants, fructose bisphosphate aldolase (EC 4.1.2.13) occurs in chloroplast, cytosol, and nucleus. Immunocytolocalization experiments with isozyme-directed antibodies indicate that both chloroplastic and cytosolic aldolase isoforms are present in the pea (Pisum sativum L.) leaf nucleus. Correspondence and reprints: Department of Biological Sciences m/c 066, University of Illinois-Chicago, 845 West Taylor, Chicago, Illinois 60607-7060, U.S.A.     

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.)

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20–56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.     

Asynchrony in larval development of the pine beauty moth, Panolis flammea, on an introduced host plant may affect parasitoid efficacy

In the United Kingdom, Panolis flammea (Den. and Schiff.) (Lepidoptera: Noctuidae) is an important pest species of the introduced lodgepole pine but not of its natural host Scots pine. The timing of P. flammea larval growth must be synchronized with its host tree if the larvae are to succeed. We collected field data during 1990 which revealed that the phenological window starts earlier in Scots pine and is shorter than that observed in lodgepole pine. The larvae are found in the field earlier and within a narrower time frame within a Scots pine forest than in a lodgepole pine forest. The larval developmental period is significantly longer on lodgepole pine than on Scots pine. The synchrony/asynchrony of P. flammea to its natural host (Scots pine) and an introduced tree (lodgepole pine) results in the parasitoids having a different impact on the larvae of the two hosts. At any one time, the host plant, caterpillars and parasitoids are more synchronous on the ancestral Scots pine than on lodgepole pine, resulting in a higher percentage of larvae in the optimal instar for parasitism at that time. In lodgepole pine, the percentage of suitable instars available to parasitoids is lower at any given time. The information presented here furthers our understanding of the possible mechanisms for the observed differential population dynamics of the insect on Scots pine and lodgepole pine in the UK. Handling editor: Robert Glinwood.     

Evaluation and first-year field testing of efficient vesicular arbuscular mycorrhizal fungi for inoculation of wetland rice seedlings

Grain yields of the rice cultivar Prakash were improved upon inoculation with Glomus intraradices and G. fasciculatum, by 11% and 8%, respectively, compared with an uninoculated control. The results indicate that the amount of phosphate fertilizer usually applied to rice may be decreased by 50%, without affecting yield, if G. intraradices is inoculated.The authors are with the Department of Agricultural Microbiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560 065, India. ing author.     

The effects of co-fungal cultures and supplementation with carbohydrate adjuncts on lignin biodegradation and substrate digestibility

The ability of three fungal strains (Pleurotus sajor-caju, Phanerochaete chrysosporium, Trametes versicolor) to decrease the lignin content and to enhance in vitro rumen digestibility of lignified spruce sawdust was assessed. In monoculture solid substrate fermentation (SSF) studies, a considerable length of time (6 weeks) elapsed before 4 to 14% lignin was degraded. In contrast, paired or multiple cultures of these fungi caused an 8 to 16% loss of native lignin within three weeks of incubation. There were also synergistic effects on total polysaccharide/hemicellulose degraded by mixed cultures. A similar observation was made for in vitro digestibility of fungal fermented samples: Total solubles (carbohydrate products) which accumulated in cultures were significantly higher in mixed cultures than in respective monocultures. In contrast, mixtures of cell free enzyme extracts of these fungi did not cause any marked reduction in lignin or cellulose content. Supplementation of wood sawdust with carbohydrate adjuncts prior to fungal treatment also led to substantial reduction in lignin content and increased substrate digestibility.F.O. Asiegbu is with the Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences, P.O. Box 7026, S-750 07 Uppsala, Sweden; A. Paterson and J.E. Smith are with the Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, G1 1XW, UK.     

Type I and Type II genes for the chlorophyll a/b-binding protein in the gymnosperm Pinus sylvestris (Scots pine): cDNA cloning and sequence analysis

A cDNA library was constructed from mRNA prepared from light-treated seedlings of Scots pine (Pinus sylvestris L.) and cDNAs for the chlorophyll a/b-binding protein LHC-II were identified using a pea gene as the heterologous probe. Three cDNA clones were sequenced. The deduced amino acid sequences of two of the genes corresponded to Type I and one to Type II LHC-II proteins which were ca. 90% homologous to their angiosperm counterparts. The transit peptides of the Scots pine preLHC-II showed features common to angiosperm transit peptides. The three cDNAs had a 70 to 75% preference for G+C in the third base position. CpG and GpC profiles and degenerate codon position bias suggested that two of the corresponding genes lie within CpG islands.     

Elicitor-induced formation of free and cell-wall-bound stilbenes in cell-suspension cultures of Scots pine (Pinus sylvestris L.)

Treatment of Pinus sylvestris L. cell-suspension cultures with an elicitor preparation from the pine needle pathogen Lophodermium seditiosum, resulted in a severalhundredto thousandfold accumulation of the stilbenes pinosylvin and pinosylvin 3-O-methyl ether in methanolic cell extracts. There was a simultaneous induction of the biosynthetic enzymes phenylalanine ammonia-lyase (E.C. 4.3.1.5.) and stilbene synthase (pinosylvin-forming, E.C. 2.3.1.146). For the first time, an incorporation of stilbenes into the cell wall fraction as well as stilbene excretion into the extracellular space was demonstrated in addition to intracellular accumulation.Abbreviations PAL phenylalanine ammonia-lyase - PS pinosylvin - PSM pinosylvin 3-O-methyl ether - STS stilbene synthase (pinosylvin-forming) This study has been supported by the Bayerisches Staatsministerium für Landesentwicklung und Umweltfragen, Fonds der chemischen Industrie, and EUROSILVA. The authors wish to thank their colleagues L. Gößl for maintaining the Scots pine cell-suspension culture, and Dr. G. Bahnweg for supplying the Lophodermium mycelia.     

Isolation, Purification, and Subcellular Localization of Isozymes of Superoxide Dismutase from Scots Pine (Pinus sylvestris L.) Needles

Two of four isozymes of superoxide dismutase (SOD) (EC 1.15.1.1) were purified from Scots pine (Pinus sylvestris L.) needles. One form was cytosolic (SOD-1) and the other was associated with chloroplasts (SOD-3). The holoenzyme molecular masses was estimated at approximately 35 kilodaltons by gel filtration. The subunit molecular weight of the dimeric enzymes was estimated to 16.5 kilodaltons (SOD-1) and 20.4 kilodaltons (SOD-3) on sodium dodecyl sulfatepolyacrylamide gels. The NH₂-terminal sequence of the pine enzymes showed similarities to other purified superoxide dismutases located in the corresponding compartment. The cytosolic form revealed two additional amino acids at position 1 and 2 at the NH₂-terminal. Both forms were cyanide- and hydrogenperoxide-sensitive and SOD-3 was found to contain approximately one copper atom per subunit, indicating that they belong to the cupro-zinc SODs. The isoelectric point was 4.9 and 4.5 for SOD-1 and SOD-3, respectively.     

Effects of phosphinotricin treatment on glutamine synthetase isoforms in Scots pine seedlings

The occurrence of GS isoenzymes has been investigated in Scots pine (Pinus sylvestris) seedlings. A transient increase of glutamine synthetase (GS, EC 6.3.1.2) activity was observed in the cotyledon whorl of plants treated with the herbicide phosphinotricin (PPT). The increase in GS activity was accompanied by a parallel accumulation of GS1 protein, which remained at high levels throughout the PPT treatment. Two-dimensional SDS-PAGE western analysis showed that pine extracts contained two GS1 polypeptides which differ in their corresponding isoelectric points. Analysis of crude extracts by ion-exchange chromatography led to the separation of two GS isoforms. The first peak (GS1-a) eluted from the columns at a low ionic strength (0.15-0.18 M KCl), whereas the second one (GS1-b) was detected at 0.5 M KCl. A detailed molecular study of both GS holoenzymes confirmed that their subunits were similar in size (about 41 kDa) but different in charge. All these data clearly demonstrate the presence of two GS1 forms in Scots pine cotyledons. Moreover, a comparison of isolated GS isoproteins with the recombinantly expressed Scots pine cytosolic subunit suggests that GS1-a corresponds to the previously characterized cDNA (pGSP114) whereas GS1-b is a minor GS isoenzyme with increased relative abundance in phosphinotricin treated plants.     

Superoxide dismutase, peroxidase, and germin-like protein activity in plasma membranes and apoplast of maize roots

Summary. The analysis of plasma membranes from maize roots by native gel electrophoresis revealed the existence of Mn-containing 120 kDa and CuZn-containing 70, 40, and 15 kDa superoxide dismutase (SOD) isoform activities. Isoelectric focusing of the plasma membranes differentiated anionic SOD isoforms with a pI of about 5 and cationic SOD isoforms at pI 8.6. Solubilization of the plasma membrane proteins further separated the cationic SOD into pI 8.6, 8.2, 8.4, and 7.2 isoforms. Double staining for both SOD and peroxidase activities showed an overlap of these activities only in the case of the high-molecular-mass (ca. 120 kDa) isoforms. High-temperature treatments demonstrated that the 120 kDa isoform was active even at 100 °C, indicating that it was a germin-like protein with superoxide-dismutating activity, different from the peroxidase with a similar molecular mass and the lower-molecular-mass CuZn-containing superoxide dismutases. These results are compared to those obtained from whole-tissue extract and apoplastic fluid. Correspondence and reprints: Maize Research Institute, POB 89-Zemun, 11081 Belgrade, Serbia and Montenegro.     

Is the Lower Shade Tolerance of Scots Pine,Relative to Pedunculate Oak,Related to the Composition of Photosynthetic Pigments?

Foliage of Scots pine (Pinus sylvestris L.) and pedunculate oak (Quercus robur L.) was collected in a mixed pine/oak forest at canopy positions differing in radiation environment. In both species, chlorophyll (Chl) a/b ratios were higher in foliage of canopy positions exposed to higher irradiance as compared to more shaded crown layers. Throughout the growing season, pine needles exhibited significantly lower Chl a/b ratios than oak leaves acclimated to a similar photon availability. Hence, pine needles showed shade-type pigment characteristics relative to foliage of oak. At a given radiation environment, pine needles tended to contain more neoxanthin and lutein per unit of Chl than oak leaves. The differences in pigment composition between foliage of pine and oak can be explained by a higher ratio of outer antennae Chl to core complex Chl in needles of P. sylvestris which enhances the efficiency of photon capture under limiting irradiance. The shade-type pigment composition of pine relative to oak foliage could have been due to a reduced mesophyll internal photon exposure of chloroplasts in needles of Scots pine, resulting from their xeromorphic anatomy. Hence, the higher drought tolerance of pine needles could be achieved at the expense of shade tolerance.     

Purification of Two Superoxide Dismutase Isozymes and Their Subcellular Localization in Needles and Roots of Norway Spruce (Picea abies L.) Trees

Two isozymes of superoxide dismutase (SOD; EC 1.15.1.1) were purified from Norway spruce (Picea abies L.) needles to apparent electrophoretic homogeneity. Purification factors were 354 for SOD I and 265 for SOD II. The native molecular mass of both purified enzymes was approximately 33 kD, as determined by gel filtration. The subunit molecular weights, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were 20,000 for SOD I and 16,000 for SOD II in the presence of 2-mercaptoethanol, and 15,800 and 15,000, respectively, in its absence. These results indicate that the native enzymes were homodimers whose subunits contained intrachain disulfide bonds. Isoelectric points determined by nondenaturing isoelectric focusing were 4.5 and 5.5 for SOD I and II, respectively. NH₂-terminal sequence analysis of the first 22 to 23 amino acids revealed 70 to 75% sequence identity with chloroplastic CuZn SODs from other plant species for SOD I, and 75% sequence identity with the cytosolic CuZn SOD from Scots pine for SOD II. SOD I was the major activity in needles and it was associated with chloroplasts. SOD II activity was dominant in roots.     

Dendrochronological analysis of 19 Norwegian grain chests

Nineteen Norwegian grain chests made of Scots pine (Pinus sylvestris L.) were analyzed by measuring tree-ring widths on photographs and scanned pictures. Seventeen of the chests were successfully dated by dendrochronology. Two of the dates are corrections of an earlier dating; the ages of these two chests were verified by radiocarbon dating. The grain chests were expected to be medieval, but four, all without carvings, proved to be post-medieval. The mean curve constructed from the dated chests matches all regional Scots pine chronologies in central and southern Norway and several from southern Sweden. All the chests were probably constructed in central Norway. Originally only sixteen chests were known, but several new ones were discovered in the course of this project.     

Observations on the subcellular distribution of the ammonium ion in maize root tissue using in-vivo 14N-nuclear magnetic resonance spectroscopy

We show that the pH dependence of the base-catalysed exchange rate of the ammonium ion provides a basis for discriminating between the cytoplasmic and vacuolar pools of ammonium in plant tissues. In vivo, ¹⁴N-nuclear magnetic resonance spectra were recorded with and without ¹H-decoupling and information on the subcellular distribution of NH ₄ ⁺ was obtained from a lineshape analysis of the ¹H-coupled spectrum. We applied this method to maize (Zea mays L.) root tissues and found that: (i), the cytoplasmic ammonium concentration was low, which was in accord with the large activity of glutamine synthetase present in the roots; and (ii), inhibition of glutamine synthetase with methionine sulphoximine increased the cytoplasmic ammonium concentration, and led to the appearance of ammonium in the xylem sap.Abbreviations GS glutamine synthetase - MSO l-methionine sulphoximine - NMR nuclear magnetic resonance - Pi inorganic phosphate On secondment to the Department of Plant Sciences, University of Oxford.We acknowledge the financial support of the Agricultural and Food Research Council. R.B. Lee also thanks the Department of Plant Sciences, University of Oxford, for hospitality.